Construction of Fluorescent Immunosensor Quenchbody to Detect His-Tagged Recombinant Proteins Produced in Bioprocess

Abstract

With the widespread application of recombinant DNA technology, many useful substances are produced by bioprocesses. For the monitoring of the recombinant protein production process, most of the existing technologies are those for the culture environment (pH, O₂, etc.). However, the production status of the target protein can only be known after the subsequent separation and purification process. To speed up the monitoring of the production process and screening of the higher-yield target protein variants, here we developed an antibody-based His-tag sensor Quenchbody (Q-body), which can quickly detect the C-terminally His-tagged recombinant protein produced in the culture medium. Compared with single-chain Fv-based Q-body having one dye, the Fab-based Q-body having two dyes showed a higher response. In addition, not only was fluorescence response improved but also detection sensitivity by the mutations of tyrosine to tryptophan in the heavy chain CDR region. Moreover, the effect of the mutations on antigen-binding was successfully validated by molecular docking simulation by CDOCKER. Finally, the constructed Q-body was successfully applied to monitor the amount of anti-SARS CoV-2 nanobody secreted into the Brevibacillus culture media.

Keywords: His-tag; fluorescent biosensor; immunoassay; recombinant protein production.

Figure 1

Protein expression and activity of anti-His scFv/Fab 3D5. (A) Structure of 3D5 Fv complexed with His6. (B) Scheme of the plasmids construction and protein expression for scFv and Fab 3D5 fragments (Figure S1). (C) Specific antigen binding of scFv/Fab 3D5 fragments and their variants detected by enzyme-linked immunosorbent assay (ELISA). Gray bars represent the signals with immobilized thioredoxin-fused LnBiT protein with His6 tag at the C-terminus. White bars represent the signals without antigen. Error bars indicate ±1 standard deviation (SD) (n = 3).

Figure 2

Construction of scFv/Fab Q-bodies labeled with maleimide dye at the N-terminus. (A) Scheme for the construction of scFv- and Fab-type Q-bodies labeled with maleimide dye at the N-terminus of H chain in scFv, and at both heavy (Fd = V_H-C_H1) and light (Lc = V_L-C_L) chains in the Fab. (B) Coomassie Brilliant Blue (CBB)-stained and (C) fluorescence images of 5-TAMRA (tetramethylrhodamine) C6 maleimide-labeled wild-type scFv/Fab Q-body and their variants.

Figure 3

Quenching efficiency of 5-TAMRA C6 labeled antibody fragments, where the fluorescence intensity of denatured state was set to 1. (A) Quenching efficiency of single labeled scFv fragment with and without WW mutation, and (B) that of double-labeled Fab Q-bodies. Gray and red bars represent the fluorescence intensity of quenched and denatured Q-bodies, respectively. The fluorescence intensities were normalized by the mean intensity of each Q-body (1 nM) in the denaturant. Error bars indicate ±1 standard deviation (SD) (n = 3).

Figure 4

His₆ peptide dose-response of the wild-type and mutant Fab Q-bodies. His₆ dose-response curves of 5-TAMRA C6- (A) or ATTO520-C2 (B) labeled Fab-type Q-body in PBST. Error bars indicate ±1 SD (n = 3).

Figure 5

Responses of Q-bodies to His-tagged protein. Dose-response curve for spiked C-terminal His6-tagged nanobody detected by TAMRA- (A) and ATTO520- (B) labeled Fab-type Q-bodies in 50% M9 culture medium after cultivation with Brevibacillus choshinensis HPD31. Error bars indicate ±1 SD (n = 3).

Figure 6

Responses of Q-bodies to His-tagged protein. His₆-tagged V_HH secreted by the transformed Brevibacillus in M9 culture was detected by TAMRA- (A) and ATTO520-(B) labeled Fab-type Q-bodies in 50% M9 culture medium. Error bars indicate ±1 SD (n = 3). Inset: CBB stained gel of V_HH-His protein in the culture medium at indicated time points.

Figure 7

The relationship between the fluorescence intensity displayed by Q-body in 50% M9 culture medium and the concentration determined by a CBB-stained SDS-PAGE gel with known amounts of bovine serum albumin (BSA) standards.

Figure 8

Docking poses of the wild-type and YW mutant.